Recombinant engineered antibodies and other recombinant human glycoproteins are typically produced by transfection into mammalian cells of genes. Available expression systems for antibodies and other complex proteins generally involve the use of vectors that integrate into the host cell genome. These vectors typically integrate at essentially random sites in the genome and the level of expression is profoundly influenced by the site of integration and the chromatin structure at the site. This fact is generally regarded as limiting the maximum expression that can be achieved from each copy of the vector and leads to a great deal of variability in the productivity of different transfectant clones. The most efficient mammalian expression systems make use of a selectable marker that can be subjected to progressively more stringent selection conditions in order to select for transfectants that express at the highest possible levels from the integrated vector sequences or have undergone gene amplification. Increasing the number of vector copies at the site of integration typically leads to concomitant increase in the productivity of the cell line. Two such amplifiable markers have been widely used: dihydrofolate reductase (DHFR; see for example U.S. Pat. Nos. 5,179,017 and 6,455,275) and glutamine synthetase (G S; Bebbington et al (1992) Bio/Technology 10, 169-175; U.S. Pat. Nos. 5,591,693; 5,827,739; 5,770,359; 5,747,308; 5,122,464).
Cell-line development using such systems is time consuming and labor-intensive because of the need to screen a large number of transfectants to identify rare clones in which the vector has integrated into a favorable site in the genome for transcription. The available gene-amplification systems also suffer from the major drawback of being limited to use in a restricted number of cell-types. In practice, the DHFR system has been largely limited to use in DHFR-minus mutant CHO cells; and GS selection has been most widely used in the NS0 mouse myeloma line.
Other cell types may have advantages in terms of growth at large scale in simple defined culture media but their use is limited by the absence of efficient gene expression systems for these cells. Furthermore, different mammalian cell types show differences in the patterns of glycosylation of the glycoproteins that they secrete or in other post-translational modifications. Altering the carbohydrate structure of antibodies, for example, can have profound effects on biological activity. Thus there is considerable interest in using cell types with particular patterns of glycosylation for generating antibodies and other recombinant proteins with different biological activities.
Therefore, there is a need for a versatile expression system that could be used in different cell types and that permits more rapid cell-line development. In addition, production of recombinant human proteins and antibodies at commercial scale is costly and there is a continuing need to develop more cost-efficient manufacturing processes. One general approach to achieving increased yields of protein from fermentors is to generate increased cell biomass and to maintain cell viability for longer periods through modifications to the culture medium, feeding of nutrients and appropriate adjustments to oxygenation rates. An extension of this approach has been to engineer cells to better withstand environmental stress and so prolong viability and secretion of product.